A Comparative Review on the Catalytic Mechanism of Nonheme Iron Hydroxylases and Halogenases

Abstract

Enzymatic halogenation and haloperoxidation are unusual processes in biology; however, a range of halogenases and haloperoxidases exist that are able to transfer an aliphatic or aromatic C–H bond into C–Cl/C–Br. Haloperoxidases utilize hydrogen peroxide, and in a reaction with halides (Cl−/Br−), they react to form hypohalides (OCl−/OBr−) that subsequently react with substrate by halide transfer. There are three types of haloperoxidases, namely the iron-heme, nonheme vanadium, and flavin-dependent haloperoxidases that are reviewed here. In addition, there are the nonheme iron halogenases that show structural and functional similarity to the nonheme iron hydroxylases and form an iron(IV)-oxo active species from a reaction of molecular oxygen with α-ketoglutarate on an iron(II) center. They subsequently transfer a halide (Cl−/Br−) to an aliphatic C–H bond. We review the mechanism and function of nonheme iron halogenases and hydroxylases and show recent computational modelling studies of our group on the hectochlorin biosynthesis enzyme and prolyl-4-hydroxylase as examples of nonheme iron halogenases and hydroxylases. These studies have established the catalytic mechanism of these enzymes and show the importance of substrate and oxidant positioning on the stereo-, chemo- and regioselectivity of the reaction that takes place.

Does Substrate Positioning Affect the Selectivity and Reactivity in the Hectochlorin Biosynthesis Halogenase?

“…Enzymatic C–Cl bond formation is a rare process in Nature, yet over the past few decades a range of haloperoxidases and halogenases have been discovered (Gribble, 2003 ; Vaillancourt et al, 2006 ; van Pée et al, 2006 ; Butler and Sandy, 2009 ; Wagner et al, 2009 ; Weichold et al, 2016 ; Agarwal et al, 2017 ; Schnepel and Sewald, 2017 ; Timmins and de Visser, 2018 ). Their catalytic mechanism, however, is still subject to controversies and understanding the fundamental details of these processes may have an impact on biotechnological advances as well as drug development.…”

An Ultrasensitive Fluorescence Assay for the Detection of Halides and Enzymatic Dehalogenation

“…To develop our new halide assay, we combined the haloperoxidase‐catalysed oxidation of halides with the use of a fluorogenic probe to detect the hypohalous acids formed (Scheme ). First, we selected a vanadium‐dependent haloperoxidase for use in the halide oxidation assay because these enzymes are very stable in the presence of hydrogen peroxide, in contrast to heme‐dependent haloperoxidases that are often inactivated by low concentrations of hydrogen peroxide …”

“…We chose the vanadium‐dependent chloroperoxidase from Curvularia inaequalis ( Ci VCPO) as it is easily expressed in Escherichia coli , stable at room temperature, and capable of oxidising chloride, bromide, and iodide to the corresponding hypohalous acids . We then selected aminophenyl fluorescein as the fluorogenic probe.…”

“…First, we selected a vanadiumdependent haloperoxidase for use in the halide oxidation assay because these enzymes are very stable in the presence of hydrogen peroxide, in contrast to heme-dependent haloperoxidases that are often inactivated by low concentrations of hydrogen peroxide. [20] We chose the vanadium-dependent chloroperoxidase from Curvularia inaequalis (CiVCPO) as it is easily expressed in Escherichia coli, stable at room temperature, and capable of oxidising chloride, bromide, and iodide to the corresponding hypohalous acids. [18a,d,e,20-21] We then selected aminophenyl fluorescein as the fluorogenic probe.…”

Recent Advances in Flavin-Dependent Halogenase Biocatalysis: Sourcing, Engineering, and Application

“…This review will focus on work carried out on flavin-dependent halogenases (electrophilic halogenases), which in contrast to heme-and vanadium-dependent halogenases, selectively derivatize small molecules, making them especially interesting for applications. Biocatalytic halogenation is carried out by enzymes called halogenases or haloperoxidase ich can be classified according to their catalytic mechanism: heme, vanadium, and flavin pendent halogenases follow an electrophilic mechanism, while non-heme iron halogenase logenate through the formation of radical intermediates, and S-adenosyl-L-methionine (SAM orinases react via a nucleophilic pathway [10]. This review will focus on work carried out on flavin pendent halogenases (electrophilic halogenases), which in contrast to heme-and vanadium pendent halogenases, selectively derivatize small molecules, making them especially interestin r applications.…”